CN108332989B - Method for analyzing high-starch-content cereal grains and observing internal structure - Google Patents
Method for analyzing high-starch-content cereal grains and observing internal structure Download PDFInfo
- Publication number
- CN108332989B CN108332989B CN201810160768.3A CN201810160768A CN108332989B CN 108332989 B CN108332989 B CN 108332989B CN 201810160768 A CN201810160768 A CN 201810160768A CN 108332989 B CN108332989 B CN 108332989B
- Authority
- CN
- China
- Prior art keywords
- dye
- starch
- dyeing
- seeds
- grains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 47
- 239000004464 cereal grain Substances 0.000 title claims abstract description 12
- 235000013339 cereals Nutrition 0.000 claims abstract description 67
- 229920002472 Starch Polymers 0.000 claims abstract description 46
- 239000008107 starch Substances 0.000 claims abstract description 46
- 235000019698 starch Nutrition 0.000 claims abstract description 42
- 238000004043 dyeing Methods 0.000 claims abstract description 37
- 239000000975 dye Substances 0.000 claims description 38
- 239000012188 paraffin wax Substances 0.000 claims description 28
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 238000002791 soaking Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 230000018044 dehydration Effects 0.000 claims description 12
- 238000006297 dehydration reaction Methods 0.000 claims description 12
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000011521 glass Substances 0.000 claims description 12
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 claims description 10
- STZCRXQWRGQSJD-GEEYTBSJSA-M methyl orange Chemical compound [Na+].C1=CC(N(C)C)=CC=C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 STZCRXQWRGQSJD-GEEYTBSJSA-M 0.000 claims description 9
- 229940012189 methyl orange Drugs 0.000 claims description 9
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 8
- 240000007594 Oryza sativa Species 0.000 claims description 8
- 235000007164 Oryza sativa Nutrition 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 235000009566 rice Nutrition 0.000 claims description 8
- 239000000853 adhesive Substances 0.000 claims description 7
- 230000001070 adhesive effect Effects 0.000 claims description 7
- 239000011546 protein dye Substances 0.000 claims description 7
- 239000001993 wax Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000007598 dipping method Methods 0.000 claims description 6
- 239000007850 fluorescent dye Substances 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 239000004744 fabric Substances 0.000 claims description 4
- 238000002844 melting Methods 0.000 claims description 4
- 230000008018 melting Effects 0.000 claims description 4
- 238000002203 pretreatment Methods 0.000 claims description 4
- 238000004018 waxing Methods 0.000 claims description 4
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- 206010040844 Skin exfoliation Diseases 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 239000002390 adhesive tape Substances 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- 239000012466 permeate Substances 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 240000006162 Chenopodium quinoa Species 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 244000062793 Sorghum vulgare Species 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 244000098338 Triticum aestivum Species 0.000 claims description 2
- 240000004922 Vigna radiata Species 0.000 claims description 2
- 235000010721 Vigna radiata var radiata Nutrition 0.000 claims description 2
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 claims description 2
- 240000001417 Vigna umbellata Species 0.000 claims description 2
- 235000011453 Vigna umbellata Nutrition 0.000 claims description 2
- 235000013305 food Nutrition 0.000 claims description 2
- 235000019713 millet Nutrition 0.000 claims description 2
- 239000001048 orange dye Substances 0.000 claims description 2
- 238000003892 spreading Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 6
- 238000003306 harvesting Methods 0.000 abstract 1
- 238000003384 imaging method Methods 0.000 abstract 1
- 238000009826 distribution Methods 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 239000011630 iodine Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000012916 structural analysis Methods 0.000 description 2
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 229940077844 iodine / potassium iodide Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000001022 rhodamine dye Substances 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
The invention discloses a method for analyzing high-starch-content cereal grains and observing internal structures, and belongs to the technical field of analysis of high-starch-content cereal grains. The method comprises the steps of fixing, rinsing, dehydrating, embedding, permeating, slicing, taking slices, imaging pretreatment, dyeing, observing and the like, is simple to operate, high in efficiency, high in tissue form integrity, high in internal component distinguishing degree, capable of observing the internal structure of the compact grain seeds, suitable for analyzing the internal structure of the grain seeds with high starch content, and particularly suitable for grain with low moisture content, compact structure and hard seeds after harvesting.
Description
Technical Field
The invention relates to a method for analyzing high-starch-content cereal grains and observing internal structures, and belongs to the technical field of analysis of high-starch-content cereal grains.
Background
The section technology is a common means for observing the internal microstructure of animal and plant samples. The animal tissue sample is sliced and stained, so that technical support is provided for the research on aspects of pathology, immunohistochemistry, in-situ hybridization and the like. In addition, with the continuous development of plant ecology and breeding technology, the slicing technology is widely applied to the research of plant breeding, identification and plant morphology establishment, and becomes an important means in the field of plant science research, so that the excellent quality of tissue slices has important significance for the research of animal and plant histochemistry.
Grains are the most important grain crops on the earth, and the application of the slicing technology in the grain aspect is mainly focused on the aspects of genetic breeding, cultivation, environment, physiology and biochemistry and the like at present and makes great progress, but the slicing technology contributes less to guiding the processing and quality judgment of the grains. With the continuous and deep research on grains, the correlation between the internal structure of grains and the quality characteristics of grains becomes the key point of research. The development of grain kernel usually needs to go through growth period, early filling period, milk stage, wax stage and complete stage, along with the development of grain, the main storage substance in the kernel, namely starch and protein group, is continuously accumulated, and the endosperm is gradually developed into a solid state with compact texture from a liquid state. Conventional grain tissue analysis is mainly based on grain slicing in the early stage of grouting, and the grain is easy to slice because the grain has high moisture content and soft texture in the early stage and is beneficial to the permeation of a fixing solution and an embedding solution. But with the further development of the seeds, the moisture in the seeds is gradually reduced, and the starch bodies and the protein bodies are continuously accumulated and tightly filled in the seeds, so that the fixing agent and the embedding agent are difficult to permeate. Meanwhile, due to the high content of starch in the grains, the grains are reduced in cohesiveness in a long dehydration process, and are hard and fragile. Meanwhile, the distribution of the components in the grains is very compact, and the conventional tissue staining method is difficult to effectively distinguish different components. Therefore, it has been difficult to achieve a dissection of the internal structure of high starch content mature kernels.
The current slicing methods that are more commonly used are paraffin slicing methods and resin slicing methods. Ogawa et al reported a method for paraffin sectioning of rice caryopsis, but the method has many steps and low inter-component discrimination. Patent CN105547793A discloses a paraffin sectioning method for mature corn seeds, patent CN104374601A discloses a resin sectioning method for mature corn seeds, and the method also uses paraffin as embedding medium for sectioning, but is different from the traditional paraffin sectioning method: the melting temperature of paraffin used in the traditional slicing method is generally between 60 and 70 ℃, the fixing, embedding, permeating and drying are all carried out at higher temperature, and for grain seeds with high starch content, the higher temperature can gelatinize starch, thereby affecting the original structure of the grain seeds. On the other hand, the traditional slicing method is mainly based on plant tissues with high water content, for example, the slicing method of caryopsis reported by Liuqi iaoquan et al, so that the fixing and permeation time is long, and for mature samples with high powder content, the sample is very hard, easy to crack and fragile due to long fixing and post-treatment time, especially in the long gradient alcohol dehydration process, so that a complete grain section is difficult to obtain. The resin slicing method is applied to structural analysis of seeds in recent years due to the characteristic of good resin permeability, but is limited due to the fact that resin cost is high, and the defects that slices are fragile, processing temperature is high, cycle is long, fluorescence observation is interfered and the like exist.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for analyzing mature grain seeds with high starch content and observing internal structures, which shortens the flaking time, effectively preserves the original structures of the internal components of the seeds, improves the slicing efficiency, and realizes structural analysis with higher discrimination degree by dry flaking and different dyeing methods. Can be applied to the analysis of the internal structure of high-starch grain grains so as to guide the processing and control of the grains.
A first object of the invention is to provide a method for pre-treatment of high starch content mature cereal grain, said method comprising moisture equilibration, fixation, dehydration, waxing, embedding, slicing and flaking; the moisture balance is to adjust the moisture of the cereal grains to 14-20%; the dehydration step is to dry the fixed grains until the moisture content is below 5 percent; the wax dipping is carried out at the temperature of 30-50 ℃.
In one embodiment of the present invention, the grains include grains having a starch content of 40% or more, such as corn, wheat, barley, quinoa, rice, millet, mung bean, red bean, soybean, and the like.
In one embodiment of the invention, the waxing process is to pre-soak the dehydrated kernels in paraffin for 1-30min, and then transfer the kernels to another paraffin for 30-120min to permeate.
In one embodiment of the present invention, the dehydration is performed by sucking the excess stationary liquid, soaking in liquid nitrogen for 1-10min, and vacuum drying for 40-60 min.
In one embodiment of the invention, the fixing is performed by soaking in a fixing solution, and placing in a vacuum environment for 30-120 min; the vacuum degree of the vacuum environment is controlled to be 0.01MPa-0.1 MPa.
In one embodiment of the invention, the fixation is performed by using a blend of one or more of formalin, paraformaldehyde, glutaraldehyde or glacial acetic acid;
in one embodiment of the present invention, the moisture balance is a moisture adjustment performed in an environment of 30 to 50 ℃ and a humidity of 60 to 100 RH%.
In one embodiment of the invention, the slicing is performed with an ultra-microtome, a cryomicrotome or a paraffin microtome.
In one embodiment of the present invention, the required slice thickness is 1-5 μm, the ultra-thin microtome is selected, the slice thickness is 5-10 μm, the frozen microtome is selected, and the slice thickness is 10-50 μm, the paraffin microtome is selected.
In one embodiment of the invention, the slide is a slide with a polymeric adhesive coated on its surface to which the sample is adhered in a flat manner.
In one embodiment of the present invention, the polymeric adhesive is a compound of one or more colloids selected from polyacrylic acid, polyvinylpyrrolidone and acacia, and the concentration of the compound is 0.01 to 1g/mL, and the glass slide is immersed in the adhesive for drying, or the adhesive thin layer is coated on the surface of the stage.
In one embodiment of the invention, the staining comprises iodine staining or fluorescent staining.
In one embodiment of the present invention, the iodine dye is specifically: taking iodine/potassium iodide solution (1% by volume fraction) and glycerol, mixing uniformly at a ratio of 1:1, dripping the dye on the section of a sample, dyeing in a dark place for 30min, then flushing off redundant dye with deionized water, repeating for 3 times, covering with a cover glass, and placing on a microscope for structure observation and polarization analysis.
In one embodiment of the present invention, the fluorescent staining is specifically: respectively adopting rhodamine, methyl red, nile red, fluorescein isothiocyanate or methyl orange dye with the concentration of 0.001-1g/mL to dye according to the sequence of protein, fat and starch; wherein, the protein is dyed by rhodamine or methyl red; the fat is dyed by Nile red; dyeing starch with fluorescein isothiocyanate or methyl orange; after each dye is dyed, repeatedly washing the dye with 30-50% ethanol for 2-3 times, and carrying out light-proof treatment in the dyeing and washing processes.
In one embodiment of the present invention, the fluorescent staining is specifically: dyeing by adopting a compound dye solution with the concentration of 0.001-1g/mL, wherein the compound dye solution contains at least two of rhodamine, methyl red, nile red, fluorescein isothiocyanate or methyl orange; when the dye is used for dyeing starch and protein, the concentration of the starch dye is controlled to be 5-20 times higher than that of the protein dye; when the dye is used for dyeing starch and fat, the concentration of the starch dye is controlled to be 2-10 times higher than that of the fat dye; when the dye is used for dyeing fat and protein, the concentration of the fat dye is 2-10 times higher than that of the protein dye; the starch dye contains Fluorescein Isothiocyanate (FITC) or methyl red; the fatty dye contains nile red; the protein dye contains rhodamine or methyl red; and repeatedly washing the dyed cloth for 2-3 times by using 30-50% ethanol, and shading the dyed cloth in the dyeing and washing processes.
In one embodiment of the present invention, the fluorescent staining is specifically: respectively weighing Fluorescein Isothiocyanate (FITC), methyl red, methyl orange, nile red and rhodamine dyes, dissolving the dyes in acetone to prepare dyes with the concentration of 0.001-1g/mL, wherein the FITC and the methyl orange are used for dyeing starch, the nile red is used for dyeing fat, and the methyl red and the rhodamine are used for dyeing protein; after dyeing, the starch generates green fluorescence, the protein generates red fluorescence, and the fat shows orange fluorescence; in order to observe the distribution of different components simultaneously, the prepared dye can be compounded; wherein, when observing starch and protein, the concentration of starch dye is controlled to be 5-20 times higher than that of protein dye, when observing starch and fat, the concentration of starch dye is controlled to be 2-10 times higher than that of fat dye, and when observing fat and protein, the concentration of fat dye is 2-10 times higher than that of protein, meanwhile, the dyeing sequence is protein, fat and starch in turn, after each dye is dyed, 50% ethanol is used for repeatedly washing for 3 times, the operation process is protected from light, and the fluorescent dyeing is mainly observed by a laser confocal microscope and a fluorescent microscope.
The second purpose of the invention is to provide a method for analyzing and observing the internal structure of the high-starch content mature cereal grains, which comprises the steps of pretreating the high-starch content mature cereal grains by using the pretreatment method and then observing.
In one embodiment of the present invention, the observation includes, but is not limited to, observation under an optical microscope, a laser confocal microscope, or a fluorescence microscope.
In one embodiment of the invention, the method comprises the steps of:
(1) sampling: selecting full and complete grains from the harvested grains, and carrying out shelling and peeling treatment;
(2) and (3) moisture balance: placing the obtained seeds in a closed container, and carrying out over-humidity adjustment; so that the moisture of the grains is kept between 14 and 20 percent;
(3) fixing: taking out the balanced seeds, soaking in the compound stationary liquid, vacuumizing, and carrying out negative pressure for 30-120 min;
(4) and (3) dehydrating: sucking off excessive fixing liquid from the fixed seeds, soaking in liquid nitrogen for 1-10min, and vacuum drying for 60 min;
(5) wax dipping: melting a certain amount of paraffin, dividing into two parts, pre-soaking the dehydrated seeds in one part for 1-30min, taking out, placing in the other part of paraffin, and infiltrating for 30-120min, wherein the paraffin soaking is carried out at 30-50 ℃;
(6) embedding: putting the soaked seeds in a mould, pouring melted paraffin into the mould, and balancing for 30-60 min;
(7) slicing: placing the embedded mould on a low-temperature platform to solidify paraffin, then using a slicer to slice, controlling the thickness of the seeds according to specific determination requirements, and continuously slicing until continuous sections appear;
(8) exhibition of slices: taking the section surface of the continuous sample by tweezers, coating polymerization adhesive and a glass slide stuck with a special adhesive tape on the surface, carrying out section fishing, and flatly sticking the sample on the glass slide and the glass slide;
(9) dewaxing: placing the glass slide with the grain section in an oven, drying at normal temperature, and dewaxing by using a dewaxing agent;
(10) and (3) observing a sample: carrying out SEM, TEM and autofluorescence pretreatment on the sample fixed on the objective table, and carrying out corresponding microscopic observation;
(11) and (3) dyeing observation: the sample treatment is carried out according to the required observation purpose, and the dyeing treatment is carried out according to different observation purposes and component heat.
The invention also provides the application of the method in the food field, such as the determination of the chalkiness and the hardness of grains, the prediction of the processing quality of grains and the product quality.
The beneficial technical effects of the invention are as follows:
the invention improves the traditional paraffin slicing method, and establishes a slicing and observing method suitable for multiple kinds of grain mature grains. The seed is subjected to moisture regulation, so that the breakage rate of the seed in the fixing process is reduced; the tissue structure of the seeds is maintained to the maximum extent through the compounding of different fixing liquids, and the slicing quality of the seeds is improved; a specific freeze-drying dehydration method for high-starch and low-moisture grains is established, the dehydration time is shortened, and the slicing efficiency is improved; the observation of the internal structures of different grain structures is realized through different slicing methods and dry-method sheet spreading; through a special fluorescent dyeing mode, the discrimination between the components is improved. In addition, the invention provides a new analysis means for the mature grain seeds with high starch content, has simple and convenient operation and low cost, provides guarantee for guiding grain processing and quality control and is beneficial to promoting the development of the grain industry in China.
Drawings
FIG. 1 is an overall sectional view of the rice slice thus produced;
FIG. 2 is a cross-sectional view of a rice grain slice taken under a scanning electron microscope;
FIG. 3 is a graph showing the distribution of starch inside rice grains after staining with iodine solution under an optical microscope;
FIG. 4 is a distribution diagram of different components inside rice grains after fluorescent staining under a confocal laser scanning microscope;
FIG. 5 is a sectional view of the whole of a sliced sheet obtained in comparative example 1;
FIG. 6 is a sectional view of a scanning electron microscope showing a section obtained in comparative example 2.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
Example 1
The specific implementation mode is as follows:
(1) sampling: selecting full and complete grains from the harvested grains, and carrying out shelling and peeling treatment;
(2) and (3) moisture balance: placing the obtained seeds in a closed container, and carrying out over-humidity adjustment; so that the moisture of the grains is kept at 16%;
(3) fixing: taking out the balanced seeds, soaking the seeds in formalin, paraformaldehyde, glutaraldehyde and glacial acetic acid (pure solution with the volume ratio of 1:1:1: 1) fixing solution, vacuumizing, and performing negative pressure of 0.5MPa for 30 min;
(4) and (3) dehydrating: taking the fixed seeds, sucking off the redundant fixing solution, soaking in liquid nitrogen for 1min, and then vacuum drying for 1 h;
(5) wax dipping: melting a certain amount of paraffin, dividing into two parts, pre-soaking the dehydrated seeds in one part for 30min, taking out, placing the seeds in the other part of paraffin, and infiltrating for 30min, wherein the paraffin soaking is carried out at 40 ℃;
(6) embedding: putting the waxed grains in a mould, pouring melted paraffin into the mould, and balancing for 1 hour;
(7) slicing: placing the embedded mould on a low-temperature platform to solidify paraffin, then using a slicer to slice, controlling the thickness of the seeds according to specific determination requirements, and continuously slicing until continuous sections appear;
(8) exhibition of slices: taking the section surface of the continuous sample by tweezers, coating polymerization adhesive and a glass slide stuck with a special adhesive tape on the surface, carrying out section fishing, and flatly sticking the sample on the glass slide and the glass slide;
(8) dewaxing: placing the glass slide with the grain section in an oven, drying at normal temperature, and dewaxing by using a dewaxing agent;
(9) and (3) observing a sample: carrying out SEM, TEM and autofluorescence pretreatment on the sample fixed on the objective table, and carrying out corresponding microscopic observation;
(10) and (3) dyeing observation: the sample is processed according to the observation purpose, and the fluorescent dyeing treatment is simultaneously carried out according to different observation purposes and component heat properties.
FIG. 1 is a cross-sectional view of the resulting slice, from which it can be seen that: the method can obtain complete and completely mature rice grain structure, has complete internal structure, no fragments and better shape retention;
FIG. 2 is an SEM image of the slices prepared by the method, and the slices prepared by the method can completely observe the distribution of the internal structure of the seed;
FIG. 3 is an LM of the slice, from which: the section prepared by the method can observe the distribution condition of starch in an iodine dyeing mode, and the discrimination of starch grains and starch bodies is high;
FIG. 4 is a CLSM map of the resulting slice, from which it can be seen that: the cell structure and the distribution of different components can be observed by the prepared section in a fluorescent dyeing mode, the left image is a fluorescent dyeing image of the amyloid, the middle image is a fluorescent dyeing image of protein, the right image is a composite dyeing image of starch and protein, the starch is in polygonal close distribution, the protein is less than the amyloid and is loosely dispersed around the amyloid, the starch and the protein in the composite image show fluorescence of different colors, the amyloid and the protein can be clearly distinguished, and the cell structure fluorescence is obvious.
Comparative example 1
The specific implementation manner is the same as that in example 1, the difference is that a fixing solution fixing method is adopted, the fixing solution is glutaraldehyde, the waxing temperature is 70 ℃, and fig. 5 is a section diagram obtained, the section structure is fragile, a complete grain structure cannot be obtained, and the starch structure in the grain is damaged.
Comparative example 2
The specific implementation manner is the same as that in example 1, the difference is that the moisture content of the grain is adjusted to be less than 5%, the fixing solution is formalin, the dehydration mode is an alcohol dehydration mode, and fig. 6 is an obtained SEM cross-sectional view, which shows that the obtained cross-section loses the original grain shape, the internal structure is fuzzy, cracks appear in different degrees, the surface structure is rough, and the original structure in the grain cannot be reflected.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A pretreatment method of mature cereal grains with high starch content is characterized by comprising the steps of water balance, fixation, dehydration, wax dipping, embedding, slicing, piece spreading and fluorescent dyeing; the moisture balance is to adjust the moisture of the cereal grain to 16%; the fixation is carried out by soaking the balanced grain seeds in formalin, paraformaldehyde, glutaraldehyde and glacial acetic acid fixing solution with the volume ratio of 1:1:1:1, vacuumizing, and carrying out negative pressure of 0.5MPa for 30 min; the dehydration step is to dry the fixed grains until the moisture content is below 5 percent; the wax dipping is carried out at the temperature of 40 ℃; the step of waxing is to pre-soak the dehydrated grains in paraffin for 1-30min, and then transferring the grains to another part of paraffin to permeate for 30-120 min; the step of dehydration is that after the redundant stationary liquid is absorbed, the fixed liquid is soaked in liquid nitrogen for 1-10min, and then vacuum drying is carried out for 40-60 min; the water balance is to regulate water at the temperature of 30-50 ℃ and the humidity of 60-100 RH%;
the fluorescent staining step is (a) or (b):
(a) respectively adopting rhodamine, methyl red, nile red, fluorescein isothiocyanate or methyl orange dye with the concentration of 0.001-1g/mL to dye according to the sequence of protein, fat and starch; wherein, the protein is dyed by rhodamine or methyl red; the fat is dyed by Nile red; dyeing starch with fluorescein isothiocyanate or methyl orange; repeatedly washing each dye with 30-50% ethanol for 2-3 times after dyeing, and carrying out light-proof treatment in the dyeing and washing processes;
(b) dyeing by adopting a compound dye solution with the concentration of 0.001-1g/mL, wherein the compound dye solution contains at least two of rhodamine, methyl red, nile red, fluorescein isothiocyanate and methyl orange; when the dye is used for dyeing starch and protein, the concentration of the starch dye is controlled to be 5-20 times higher than that of the protein dye; when the dye is used for dyeing starch and fat, the concentration of the starch dye is controlled to be 2-10 times higher than that of the fat dye; when the dye is used for dyeing fat and protein, the concentration of the fat dye is 2-10 times higher than that of the protein dye; the starch dye contains fluorescein isothiocyanate or methyl orange; the fatty dye contains nile red; the protein dye contains rhodamine or methyl red; and repeatedly washing the dyed cloth for 2-3 times by using 30-50% ethanol, and shading the dyed cloth in the dyeing and washing processes.
2. The method of claim 1, wherein the grain comprises corn, wheat, barley, quinoa, rice, millet, mung bean, red bean, or soybean.
3. A method of high starch content mature grain kernel profiling and internal structure observation characterized by applying the method of claim 1 or 2 for pre-treatment prior to observation.
4. The method of claim 3, wherein the observing is under an optical microscope.
5. The method of claim 3, wherein the observing is under a confocal laser microscope or a fluorescence microscope.
6. A method according to claim 3, characterized by the steps of:
(1) sampling: selecting full and complete grains from the harvested grains, and carrying out shelling and peeling treatment;
(2) and (3) moisture balance: placing the obtained seeds in a closed container, and carrying out humidity adjustment; so that the moisture of the grains is kept at 16%;
(3) fixing: taking out the balanced seeds, soaking in the compound stationary liquid, vacuumizing, and performing negative pressure for 30 min;
(4) and (3) dehydrating: sucking off excessive fixing liquid from the fixed seeds, soaking in liquid nitrogen for 1-10min, and vacuum drying for 60 min;
(5) wax dipping: melting a certain amount of paraffin, dividing into two parts, pre-soaking the dehydrated seeds in one part for 1-30min, taking out, placing in the other part of paraffin, and infiltrating for 30-120min, wherein the paraffin soaking is carried out at 40 ℃;
(6) embedding: putting the soaked seeds in a mould, pouring melted paraffin into the mould, and balancing for 30-60 min;
(7) slicing: placing the embedded mould on a low-temperature platform to solidify paraffin, then using a slicer to slice, controlling the thickness of the seeds according to specific determination requirements, and continuously slicing until continuous sections appear;
(8) exhibition of slices: fishing out the slices in the step (7), picking up the continuous sample slices by tweezers, adhering the continuous sample slices on a glass slide with the surface coated with polymerization adhesive or a special adhesive tape, and placing the glass slide on an object stage;
(9) dewaxing: placing the glass slide with the grain section in an oven, drying at normal temperature, and dewaxing by using a dewaxing agent;
(10) and (3) observing a sample: carrying out SEM, TEM and autofluorescence pretreatment on the sample fixed on the objective table, and carrying out corresponding microscopic observation;
(11) and (3) dyeing observation: processing the sample according to the required observation purpose, and dyeing according to different observation purposes and component characteristics.
7. Use of the method according to any one of claims 1 to 6 in the food field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810160768.3A CN108332989B (en) | 2018-02-27 | 2018-02-27 | Method for analyzing high-starch-content cereal grains and observing internal structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810160768.3A CN108332989B (en) | 2018-02-27 | 2018-02-27 | Method for analyzing high-starch-content cereal grains and observing internal structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108332989A CN108332989A (en) | 2018-07-27 |
| CN108332989B true CN108332989B (en) | 2020-12-29 |
Family
ID=62929896
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810160768.3A Active CN108332989B (en) | 2018-02-27 | 2018-02-27 | Method for analyzing high-starch-content cereal grains and observing internal structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108332989B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110487615B (en) * | 2019-08-29 | 2022-05-17 | 沈阳农业大学 | Compound fluorescent dyeing method for identifying clubroot of cruciferous plants |
| CN113567482A (en) * | 2021-07-16 | 2021-10-29 | 江南大学 | Method for monitoring internal component structure and distribution of grains in cooking process |
| CN114088496B (en) * | 2021-11-25 | 2023-06-20 | 内蒙古工业大学 | Device for preparing transmission electron microscope powder sample |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630421B (en) * | 2013-12-03 | 2016-03-23 | 沈阳农业大学 | A kind of paraffin section method for making of Chinese herbaceous peony Mature Embryos Among |
| CN105699142A (en) * | 2016-02-18 | 2016-06-22 | 石河子大学 | Paraffin section making method for close-texture hard plant materials |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914461B (en) * | 2012-10-24 | 2015-05-13 | 山东省花生研究所 | Paraffin sectioning method for peanut seeds |
| CN102967493B (en) * | 2012-10-27 | 2015-04-08 | 山西农业大学 | Rapid paraffin sectioning method for plant tissue |
| CN104374601B (en) * | 2014-12-08 | 2018-02-16 | 扬州大学 | A kind of resin slicer preparation method of kernel maturity seed |
| CN104849110B (en) * | 2015-04-03 | 2018-03-16 | 成都大学 | A kind of paraffin section method of plant tissue |
| CN105547793B (en) * | 2016-01-13 | 2018-01-30 | 扬州大学 | A kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method |
-
2018
- 2018-02-27 CN CN201810160768.3A patent/CN108332989B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630421B (en) * | 2013-12-03 | 2016-03-23 | 沈阳农业大学 | A kind of paraffin section method for making of Chinese herbaceous peony Mature Embryos Among |
| CN105699142A (en) * | 2016-02-18 | 2016-06-22 | 石河子大学 | Paraffin section making method for close-texture hard plant materials |
Non-Patent Citations (4)
| Title |
|---|
| Structural properties and digestion of green banana flour as a functional ingredient in pasta;Zheng, Zeqi et al.;《FOOD & FUNCTION》;20161231;第7卷(第2期);771-780 * |
| 溴氯酚蓝染料结合法测定体液白蛋白;丁玉珠 等;《上海医学检验杂志》;19931231;第8卷(第2期);107-108 * |
| 牛血清白蛋白与甲基橙结合反应的研究;魏永巨 等;《高等学校化学学报》;19961130;第17卷(第11期);1687-1692 * |
| 石蜡切片中同时显示植物细胞内淀粉、蛋白质和脂肪的方法;张杏辉;《生物技术》;19951231;第5卷(第3期);45-46 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108332989A (en) | 2018-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108332989B (en) | Method for analyzing high-starch-content cereal grains and observing internal structure | |
| Konarska | The structure of the fruit peel in two varieties of Malus domestica Borkh.(Rosaceae) before and after storage | |
| CN109855936B (en) | Preparation method of plant tissue section with complete tissue and favorable microscopic observation | |
| CN104849110B (en) | A kind of paraffin section method of plant tissue | |
| Ebadi et al. | Effect of low temperature near flowering time on ovule development and pollen tube growth in the grapevine (Vitis vinifera L.), cvs Chardonnay and Shiraz | |
| CN105973673A (en) | Paraffin sectioning method for eucalyptus tissue | |
| CN107702959A (en) | A kind of paraffin section method for vegetable material serial dehydration | |
| CN105699142A (en) | Paraffin section making method for close-texture hard plant materials | |
| CN109738249A (en) | A kind of production method of paraffin section that observing Process of Flower Bud Differentiation | |
| CN111257089B (en) | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid | |
| Grange et al. | Seedcoat structure and oxygen-enhanced environments affect germination of triploid watermelon | |
| Vu et al. | Structure and chemical composition of wild soybean seed coat related to its permeability | |
| CN108680418B (en) | Dyeing liquid and method for quickly dyeing cruciferous crop pollen | |
| CN107132097B (en) | Slicing method suitable for observing anatomical structures of flower organs of rubber trees | |
| CN110073760B (en) | Pre-wetting solution, staining solution and method for rapidly determining vitality of Iris tenuifolia seeds | |
| Karahara et al. | High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy | |
| CN110926907A (en) | Method suitable for making equine animal ovary paraffin section | |
| CN107702966A (en) | A kind of method for making water spinach root knot megabacterium paraffin section | |
| WO1997043418A1 (en) | Globulin protein 11s, usable as a seed impregnation marker during germination. | |
| CN107202720B (en) | A kind of paraffin section method of pomegranate seed | |
| Sylvester et al. | Light microscopy I: dissection and microtechnique | |
| Shumborski et al. | Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development | |
| CN111982629B (en) | Ultrathin frozen slicing method for edible fungus tissue cells | |
| Gao et al. | Structural characteristics of the mature embryo sac of Camellia oleifera | |
| Neya et al. | Ageing increases the sensitivity of neem (Azadirachta indica) seeds to imbibitional stress |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |